Thermophilic fungal expression system

ABSTRACT

The present invention relates to recombinant thermophilic host cells comprising a nucleic acid sequence encoding a heterologous protein, and a method of producing recombinant protein utilizing same. The recombinant hosts of the present invention provide a better morphology in tank fermentations than many known fungal host cells, such as Aspergillus, which morphology results in lower viscosity levels, and therefore improved productivity.

This is a divisional application of co-pending application Ser. No.08/278,473, filed July 20, 1994 the contents of which are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to host cells useful in the production ofrecombinant proteins. In particular, the invention relates tothermophilic fungal host cells which can be used in the expression ofrecombinant proteins, especially enzymes.

BACKGROUND OF THE INVENTION

The use of recombinant host cells in the expression of heterologousproteins has in recent years greatly simplified the production of largequantities of commercially valuable proteins, which otherwise areobtainable only by purification from their native sources. Currently,there is a varied selection of expression systems from which to choosefor the production of any given protein, including prokaryotic andeukaryotic hosts. The selection of an appropriate expression system willoften depend not only on the ability of the host cell to produceadequate yields of the protein in an active state, but also to a largeextent may be governed by the intended end use of the protein.

Although mammalian and yeast cells have been the most commonly usedeukaryotic hosts, filamentous fungi have now begun to be recognized asvery useful as host cells for recombinant protein production. Certainspecies of the genus Aspergillus have been used effectively as hostcells for recombinant protein production. Furthermore, there are oftenproblems with the formation of too dense aggregates of mycelium anduneven distribution, which also results in starvation for nutrients andan unproductive situation. The species Aspergillus nidulans has beenreported to be transformed with recombinant plasmids (Ballance, et al.Biochem. Biophys. Res. Comm. 112: 284-289, 1983), but transformation wasfound to occur at fairly low frequency. Both Aspergillus niger andAspergillus oryzae have also been described as being useful inrecombinant production of heterologous proteins. Although these speciesare currently routinely used in recombinant protein production, they arenot without their drawbacks. In particular, their growth morphology infermentors are not optimum for fermentation, as viscosity tends tobecome rather high as biomass increases. Increased viscosity limits theability to mix and aerate the fermentation culture, leading to oxygenand nutrient starvation of the mycelia, which therefore become inviableand unproductive. Furthermore, there are often problems with theformation of too dense aggregates of mycelium, and uneven distribution,which also results in starvation for nutrients. Therefore, forcommercial purposes, there continues to be a need for fungal hosts whichare capable of use in expression of recombinant proteins but whichexhibit satisfactory growth characteristics, such as rapid growth andlow viscosity, thereby enhancing productivity in fermentors.

SUMMARY OF THE INVENTION

The present invention provides novel recombinant fungal host cells,which cells exhibit growth characteristics particularly well suited foruse in production of heterologous proteins in fermentors. The host cellsof the invention are capable of rapid growth, exhibit low viscosity at agiven biomass concentration and result in an evenly dispersed myceliumwith a loose enough structure to allow sufficient diffusion of nutrientsto all parts of the mycelium. In particular, the host cells of theinvention are thermophilic fungi, which under equivalent fermentationconditions, for example, in batch fermentation at pH 5, in a salt, yeastextract medium containing 100 g/l glucose, produce viscosity values,measured, for example, in pascal, which are less than 80%, preferablyless than 50%, and most preferably less than 30% the viscosity valuesproduced under the same conditions at the same biomass by Aspergillusoryzae; the preferred fungal hosts produce a homogeneous, loosestructure of the mycelium. Most preferably, the fungal host cells areselected from the group consisting of Thielavia sp., Thermoascus sp.,Myceliophthora sp., and Sporotrichum sp. The invention thereforeprovides recombinant host cells, as defined above, comprising a nucleicacid fragment encoding a heterologous protein (which is hereinunderstood also to encompass peptides), which protein can be expressedby the host cell. The invention further provides a method for productionof heterologous proteins comprising culturing a host cell of theinvention under conditions conducive to the expression of theheterologous protein of interest, and recovering the protein fromculture.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the viscosity in batch fermentation, measured inpascal, and extrapolated to 30 g/l, of a number of thermophilic fungicompared with that of Aspergillus oryzae.

FIG. 2 shows the southern hybridization analysis of total DNA isolatedfrom untransformed and xylanase transformants of thermophilic strains. A1.2 kb HindIII-XhoI fragment of Humicola insolens xylanase cDNA is usedas the probe. Lane 1: Myceliophthora thermophila untransformed; Lane 2:Myceliophthora thermophila transformant #5; Lane 3: Myceliophthorathermophila transformant #11; Lane 4: Acremonium alabamenseuntransformed; Lane 5: Acremonium alabamense transformant #5; Lane 6:Acremonium alabamense transformant #8; Lane 7: Sporotrichumcellulophilum untransformed; Lane 8: Sporotrichum cellulophilumtransformant #6; Lane 9: Sporotrichum cellulophilum transformant #7.

FIG. 3 illustrates the increase in biomass over time for Thielaviaterrestris 373, and a nonsporulating mutant of the same strain. Thearrow indicates the point in time at which the 373 strain begins tosporulate.

DETAILED DESCRIPTION OF THE INVENTION

The commercial use of any recombinant protein largely depends on theability to achieve efficient production in large-scale fermentation.Productivity is limited by a number of factors in industrialfermentations of fungi. The common problems are related to relativelyhigh viscosity compared to unicellular organisms, such as Saccharomycescerevisiae and Bacillus sp., and the often very heterogeneousdistribution of mycelium in dense aggregates, that cause a major part ofthe mycelium to starve, due to lack of O₂ and/or nutrient diffusion toall cells. The high viscosity reduces the oxygen transfer rate that canbe reached in the fermentor, which in turn adversely effects the overallenergy the cells can produce, thereby leading to lower concentration ofobtainable productive biomass and lower final product yield or longerfermentation times. It can therefore be seen that simply increasingbiomass is not, without the proper morphology that leads to lowviscosity, adequate to increase yield in fermentation. There must be anincrease in productive biomass in order for any advantages to beobtained.

It has now been discovered that a number of thermophilic fungiunexpectedly exhibit certain growth characteristics which render themsuitable for culturing in fermentors. Attempts to identify fungi withuseful growth characteristics begins as a random screening of ataxonomically heterogeneous group of thermophilic fungi under a varietyof culture conditions. Shake flask evaluations focus in large part ondetermining whether the candidate strains produce large quantities ofextracellular proteins and/or proteases, each of which is an undesirablecharacteristic in a cell to be used for recombinant protein production.Also observed, however, is the growth and morphology of each strain.Initially, a rapid growth rate, combined with a loose, homogeneousdistribution of mycelia, with neither large pellets nor aggregatesformed in culture, is considered indicative of a good candidate for usein the fermentor. Based on this initial screening, strains of thefollowing species are selected for testing in fermentors: Thermoascusthermophilus, Mucor pusilus, Myceliophthora thermophila, Thielaviaterrestris, Acremonium alabamense (the imperfect form of Thielaviaterrestris), Talaromyces emersonii, and Sporotrichum cellulophilum.

The candidate strains are tested in six 100 g/l glucose batchfermentation runs and analyzed for viscosity, with Aspergillus oryzae asthe control. Mucor pusilus produces a large cake of mycelia, andTalaromyces strains produces high levels of protease, and therefore, aredropped from further studies. The data for the remaining strains,provided in Table 1, illustrates that the viscosity levels of thethermophiles tested are substantially (i.e., at least about 50%) lowerthan the levels observed with Aspergillus oryzae at equivalentbiomasses. An extrapolation of these data, comparing the strains at 30g/l of biomass, is shown in FIG. 1. Based strictly on the observationson viscosity, Thielavia terrestris appears to have the most favorableprofile. The mycelia in this species are homogeneously dispersed,forming a loose structure of close, highly branched mycelia.Myceliophthora is similar to Thielavia but has an elongated and lessbranched growth form, resulting in a somewhat higher viscosity than seenwith Thielavia. Both Thermoascus and Sporotrichum also exhibit a usefulmorphology and very little viscosity.

In addition to exhibiting a useful morphology, the candidate host cellmust of course be transformable and capable of expressing heterologousprotein. Although thermophilic fungi, e.g., Humicola grisea var.thermoidea, have previously been transformed (Allison et al., Curr.Genet. 21:225-229, 1992), the expression of heterologous proteins in arecombinant fungal cell has not been reported. Therefore, it wasinitially unclear that these thermophiles would ultimately prove usefulat all in recombinant heterologous protein production. Surprisingly,however, as shown in the Examples below, the standard Aspergillustransformation protocols (as described in, for example, Christiansen etal., Bio/Technol. 6: 1419-1422) can be used to transform a majority ofthe strains tested. Thus, the thermophilic fungi of the presentinvention provide the requisite properties for use as host cells inrecombinant protein production in fermentors, both with regard totransformability and advantageous morphology.

The use of thermophilic fungi as host cells provides other advantages aswell. In addition to the lower viscosity observed in culture of thesefungi, the higher temperature at which they are grown is conducive to amore rapid growth rate in some species than is seen withnon-thermophiles. This in turn leads to a more rapid accumulation ofbiomass, which results in a relatively short fermentation cycle. Also,in continuous fermentation, the combination of the higher temperatureswith the lower pH which these fungi favor provides conditions in whichrisk of contamination is significantly reduced.

The present invention encompasses any thermophilic fungus which meetsthe viscosity requirements as defined above during fermentation. By"thermophilic fungus" is meant any fungus which exhibits optimum growthat a temperature of at least about 40° C., preferably between 40°-50° C.This includes, but is not limited to, the thermophilic members of thegenera Acremonium, Corynascus, Thielavia, Myceliophthora, Thermoascus,Sporotrichum, Chaetomium, Ctenomyces, Scytalidium, and Talaromyces. In apreferred embodiment, the thermophile is selected from the groupconsisting of strains of Thermosascus thermophilus, Myceliophthorathermophila, Sporotrichum cellulophilum, and Thielavia terrestris. Itwill be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. For example, the imperfect form of Thielavia terrestrisis known as Acremonium alabamense, and Myceliophthora thermophila isThielavia heterothallica. Further examples of taxonomic equivalents andother useful species can be found, for example, in Cannon,Mycopathologica 111: 75-83, 1990; Moustafa et al., Persoonia 14:173-175, 1990; Stalpers, Stud. Mycol. 24, 1984; Upadhyay et al.,Mycopathologia 87: 71-80, 1984; Subramanian et al., Cryptog. Mycol. 1:175-185, 1980; Guarro et al., Mycotaxon 23: 419-427, 1985; Awao et al.,Mycotaxon 16: 436-440, 1983; von Klopotek, Arch. Microbiol. 98:365-369,1974; and Long et al., 1994, ATCC Names of Industrial Fungi, ATCC,Rockville, Md. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

As the results presented in the examples show, several isolates of eachspecies possess the morphology required to make them useful infermentation. It is also shown that the ability to be transformed is notlimited to a single thermophilic species. Thus, it is understood thatthe utility is not limited to a single isolate or strain, but rather isa characteristic of a group of species. Those skilled in the art willrecognize that other strains or isolates of these species can also beused in expression of heterologous expression. Many strains of eachspecies are publicly available in the collections of the American TypeCulture Collection (ATCC) 12301 Parklawn Drive, Rockville Md. 20852;Agricultural Research Service Culture Collection (NRRL) 1815 NorthUniversity Street, Peoria, Ill. 61604; Fungal Genetics Stock Center(FGSC), Kansas; Deutsche Sammlung yon Mikroorganismen und Zellkulturen(DSM), Mascheroder Weg 1B, D-3300 Braunschweig, Germany; Institute ofApplied Microbiology (IAM), Tokyo University 1-1,1-Chome, Yayoi,Bunkyo-ku, Tokyo 113, Japan; Institute for Fermentation (IFO), 17-85Juso-honmachi 2-chome, Yodogawaku, Osaka 532, Japan; and Centraal Bureauvoor Schimmelcultures (CBS), Oosterstraat 1, 3740 AG Baarn, Netherlands,and are also available in the culture collection of Novo NordiskBiotech, Davis, Calif.

Suitability of other thermophilic fungal hosts for use in fermentors canreadily be determined by the methods described in the followingexamples. Briefly, candidate fungi are cultured on standard growthmedium such as salts/yeast extract, soya, potato protein, or any mediumsupplemented with glucose or other appropriate carbon source. Thefermentation is carried out at a pH of about 4-7 and at a temperature offrom about 37°-50° C., preferably at about 42°-46° C. It will of coursebe recognized that the temperature of the control fermentation should bethat which is optimum for the control strain; for A. oryzae, this isabout 32°-36° C. It is possible to identify qualitatively those strainswhich will be useful for fermentation by visual inspection of mycelialmorphology, in shake flasks; useful strains will show a loose,homogeneous arrangement of mycelium with many branching points.Confirmation of utility is best determined in fermentors, by evaluatingactual viscosity of the culture medium at various time points in thefermentation. Viscosity determination can be made by any means known inthe art, e.g., Brookfield rotational viscometry (defined or unlimitedshear distance and any type of spindle configuration), kinematicviscosity tubes (flow-through tubes), falling ball viscometer orcup-type viscometer. Preferably, in the evaluation, a strain of A.oryzae is included as a control with which the viscosity of thecandidate strain is compared. The preferred host cells exhibit about 80%or less of the viscosity level produced by an A. oryzae strain underidentical fermentation conditions, preferably about 50% or less, andmost preferably about 30% or less.

The skilled artisan will also recognize that the successfultransformation of the host species described herein is not limited tothe use of the vectors, promoters, and selection markers specificallyexemplified. Generally speaking, those techniques which are useful intransformation of A. oryzae, A. niger and A. nidulans are also usefulwith the host cells of the present invention. For example, although theamdS selection markers are preferred, other useful selection markersinclude, but are not limited to, the argB (A. nidulans or A. niger),trpC (A. niger or A. nidulans), pyrG (A. niger or A. nidulans), sC(selenate resistance) or glufosinate resistance (A. oryzae) markers, ortheir equivalents from other species. The promoter may be any DNAsequence that shows strong transcriptional activity in these species,and may be derived from genes encoding both extracellular andintracellular proteins, such as amylases, xylanases, glucoamylases,proteases, lipases, cellulases and glycolytic enzymes. Such suitablepromoters may be derived from genes for A. oryzae TAKA amylase,Rhizomucor miehei aspartic proteinase, A. niger glucoamylase, A. nigerneutral α-amylase, A. niger acid stable α-amylase, and Rhizomucor mieheilipase. Examples of promoters from genes for glycolytic enzymes are TPI,ADH, and PGK. The promoter may also be a homologous promoter, i.e., thepromoter for a gene native to the host strain being used. A usefulpromoter according to the present invention is the A. oryzae TAKAamylase promoter. The TAKA amylase is a well-known α-amylase (Toda etal., Proc. Japan Acad. 58 Ser. B.: 208-212, 1982). The promoter sequencemay also be provided with linkers for the purpose of introducingspecific restriction sites facilitating ligation of the promotersequence with the gene of choice or with a selected signal peptide orpreregion. Terminators and polyadenylation sequences may also be derivedfrom the same sources as the promoters. Enhancer sequences may also beinserted into the construct.

To avoid the necessity of disrupting the cell to obtain the expressedproduct, and to minimize the amount of possible degradation of theexpressed product within the cell, it is preferred that the product besecreted outside the cell. To this end, in a preferred embodiment, thegene of interest is linked to a preregion such as a signal or leaderpeptide which can direct the expressed product into the cell's secretorypathway. The preregion may be derived from genes for any secretedprotein from any organism, or may be the native preregion. Among usefulavailable sources for such a preregion are a glucoamylase or an amylasegene from an Aspergillus species, an amylase gene from a Bacillusspecies, a lipase or proteinase gene from Rhizomucor miehei, the genefor the α-factor from Saccharomyces cerevisiae, or the calf prochymosingene. Most preferably the preregion is derived from the gene for A.oryzae TAKA amylase, A. niger neutral α-amylase, A. niger acid stableα-amylase, B. licheniformis α-amylase, the maltogenic amylase fromBacillus NCIB 11837, B. stearothermophilus α-amylase, or B.licheniformis subtilisin. An effective signal sequence is the A. oryzaeTAKA amylase signal, the Rhizomucor miehei aspartic proteinase signaland the Rhizomucor miehei lipase signal. As an alternative, thepreregion native to the gene being expressed my also be used.

The gene for the desired product functionally linked to promoter andterminator sequences may be incorporated in a vector containing theselection marker or may be placed on a separate vector or plasmidcapable of being integrated into the genome of the host strain. Thevector system may be a single vector or plasmid or two or more vectorsor plasmids which together contain the total DNA to be integrated intothe genome. Vectors or plasmids may be linear or closed circularmolecules. According to a preferred embodiment of the present invention,the host is transformed with two vectors, one including the selectionmarker and the other comprising the remaining heterologous DNA to beintroduced, including promoter, the gene for the desired protein andtranscription terminator and polyadenylation sequences.

The present host cell species can be used to express any prokaryotic oreukaryotic heterologous peptide or protein of interest, and ispreferably used to express eukaryotic peptides or proteins. The speciesThielavia terrestris is particularly useful in that it recognized ashaving a safe history. Of particular interest for these species is theiruse in expression of heterologous proteins, especially fungal enzymes.The novel expression systems can be used to express enzymes such ascatalase, laccase, phenoloxidase, oxidase, oxidoreductases, cellulase,xylanase, peroxidase, lipase, hydrolase, esterase, cutinase, proteaseand other proteolytic enzymes, aminopeptidase, carboxypeptidase,phytase, lyase, pectinase and other pectinolytic enzymes, amylase,glucoamylase, α-galactosidase, β-galactosidase, α-glucosidase,β-glucosidase, mannosidase, isomerase, invertase, transferase,ribonuclease, chitinase, mutanase and deoxyribonuclease. It will beunderstood by those skilled in the art that the term "fungal enzymes"includes not only native funsal enzymes, but also those fungal enzymeswhich have been modified by amino acid substitutions, deletions,additions, or other modifications which may be made to enhance activity,thermostability, pH tolerance and the like.

The present host cells may also be used in recombinant production ofproteins which are native to the host cells. Examples of such useinclude, but are not limited to, placing a thermophile's native proteinunder the control of a different promoter to enhance expression of theprotein, to expedite export of a native protein of interest outside thecell by use of a signal sequence, or to increase copy number of aprotein which is normally produced by the subject host cells. Thus, thepresent invention also encompasses, within the scope of the term"heterologous protein", such recombinant production of homologousproteins, to the extent that such expression involves the use of geneticelements not native to the host cell, or use of native elements whichhave been manipulated to function in a manner not normally seen in thehost cell.

As noted above, Thielavia terrestris is, because of its excellentmorphology, among the preferred species for use in recombinant proteinproduction. However, this species, in submerged culture, is also subjectto loss of biomass, by virtue of sporulation, under conditions oflimitation of growth (either glucose or oxygen). Specifically, the largeamounts of mycelia produced during early stages of fermentation mayrapidly disappear and large quantities of spores appear. Since themycelia are the source of production of the recombinant proteins,avoiding sporulation in culture is desirable. To this end, anon-sporulating mutant of Thielavia is isolated. Spores are exposed toultraviolet light and cultivated for five days. The mycelia are thenspread on plates and incubated for 24 hours. The eight largest coloniesare chosen, on the assumption that mycelial fragments grow faster thanspores which first have to germinate, and tested in shake flasks. Twoout of the eight colonies show no sporulation in submerged culture,showing this to be a viable approach in the production ofnon-sporulating strains if the need arises.

The present invention will be further illustrated by the followingnon-limiting examples.

I. Shake Flask Evaluation of thermophiles

ASPO4 medium having the following composition, with variable carbonsource, is used in shake flasks:

    ______________________________________                                        Yeast extract         2     g/l                                               MgSO.sub.4.7H.sub.2 O 1     g/l                                               CaCl.sub.2            1     g/l                                               KH.sub.2 PO.sub.4     5     g/l                                               Citric acid           2     g/l                                               Trace metals*         0.5   ml/l                                              Urea                  1     g/l                                               (NH.sub.4).sub.2 SO.sub.4                                                                           2     g/l                                               ______________________________________                                         *contains 14.3 g/l ZnSO.sub.4.7H.sub.2 O, CuSO.sub.4.5H.sub.2 O, 0.5 g/l      NiCl.sub.2.6H.sub.2 O, 13.8 g/l FeSO.sub.4.7H.sub.2 O, 8.5 g/l                MnSO.sub.4.H.sub.2 O, and 3 g/l citric acid                              

Propylene shake flasks (100 or 500 ml) without baffles are used, shakenat 200 rpm at a temperature of 42°-44° C., pH 4.5.

A number of thermophilic strains are screened in shake flasks for thefollowing characteristics: (1) vigor of growth; (2) protease production;(3) secreted proteins; and (4) mycelial morphology. To determineprotease activity, supernatant from the culture broth of each is spun at2500 rpm for 5 minutes, and used in the casein clearing plate assay,which determines the levels of proteases produced by various fungalspecies being evaluated as potential candidates for recombinant proteinexpression.

The casein plate clearing assay is conducted as follows. The platemedium is composed of 20 g/l skim milk, 20 g/l agarose, and 0.2Mcitrate-phosphate buffer for tests run at pH 5 and pH 7, and glycineNaOH buffer for tests run at pH 9. Milk powder is mixed with 100 ml ofbuffer and kept at 60° C. Agarose is mixed with 400 ml of buffer andautoclaved 5 minutes. After slight cooling, the warm milk mixture isadded, and the mixture inverted gently 2-3 times to mix. The medium ispoured into 150 mm plates using 50-70 ml per plate and stored at 5° C.until use.

Just prior to use, twelve holes per plate are made in the agar. 25 μl ofsupernatant from fermentation of each strain is added to one plate ofeach pH and incubated overnight at 37° C. To pH 9 plates, 0.5M glacialacetic acid is added to precipitate casein and allow visualization ofany clear zones. Each plate is then evaluated on clear zone size (i.e.,from no zone to >2 cm in diameter) and zone type (i.e., clear, opaque orboth types).

The supernatants of each culture are also used to evaluate the strains'extracellular protein production. Novex 8-16% SDS polyacrylamidegradient gels, prepared according to manufacturer's instructions, areused to assess the protein profile. A 40 μl (48 hour) sample of culturesupernatant is mixed with 10 μl of 5× dissociation buffer (dissociationbuffer=4 ml 1M Tris-HCl, pH 6.8, 1 g SDS, 617 mg dithiothreitol, andsterile distilled water to 10 ml), and glycerol/bromophenol blue (10-20mg added to about 10 ml of 80-90% glycerol, and placed in boiling waterfor 1-2 hours to dissolve), boiled for 5 minutes, cooled, loaded and runat 120 V until the bromphenol blue tracking dye reaches the bottom ofthe gel. The gels are stained with Coomasie brilliant blue. Thoseisolates showing large numbers of bands are considered less suitable aspotential new hosts.

Growth is evaluated qualitatively, on a + to +++ scale. Morphology isranked as follows: 1--long close interacting hyphae with very littlebranching; 2--many conidia spores, with very pronounced branching;3--thin, long, straight hyphae with some branching; 4--thick, short,irregular hyphae with lots of branching; 5--loose mycelium with verypronounced branching; 6--loose branched mycelium with very homogeneousmycelium distribution; 7--long close interacting hyphae with somebranching. Morphologies 5 and 6 are considered the most desirable.

Five experiments in shake flasks are conducted, in which the identityand amount of carbon source are varied, as follow: (1) maltodextrin 10g/l; (2) glucose 10 g/l; (3) maltodextrin 20 g/l; (4) 10 g/l Avicell+1g/l glucose for induction of cellulases!; (5) 30 g/l maltodextrin+10 g/lglucose. The isolates of the species tested, in one or more of theexperiments, are: Talaromyces emersonii, T. byssochlamydoides, Thielaviaterrestris, Thermoascus thermophilus, T. aurantiacus, Malbrancheasulfurea, Melanocarpus albomyces, Sporotrichum cellulophilum, Acremoniumalabamense, Humicola grisea var. thermoidea, Mucor pusilus,Myceliophthora thermophila, and Scytalidium thermophilum. Several ofthese strains exhibit useful morphology and vigorous growth under one ormore of the experimental conditions defined above. Under the conditionstested, none of the strains tested secrete high (>1 g/l) levels of anyprotein, and are considered to have a clean protein background. Severalof the strains show high protease activity (e.g., Talaromyces emersonii,Talaromyces byssochlamydoides) and some grow very poorly under thetested growth conditions (e.g., Malbranchea sulfurea). None of these aretested further.

In the course of this evaluation, the extent of sporulation in submergedculture and on plates is determined. The ability to sporulate on platesis virtually essentials for in a useful host cell. Of the species ofinterest, neither Thielavia terrestris nor Myceliophthora thermophilashow any spores on normal fungal agar plates. Methods for sporulationare then developed for these two species. For Myceliophthora, myceliumis first grown on potato dextrose agar (PDA; Difco) plates at 37° C. for48 hours, then grown overnight at 50° C., then grown an additional 24-48hours at 37° C. Temperature stress apparently triggers sporulation inthis species.

With Thielavia, sporulation on plates can be induced by first incubatingmycelium on PDA plates for three days in the normal atmosphere of a 37°C. incubation room. The culture is then placed in a 1 liter Pyrex®beaker that is flushed with N₂ gas, sealed with plastic wrap, andreturned to the 37° C. room for 48 hours. The plate is removed from thebeaker to the normal atmosphere of the room and left to incubate for oneweek. The plate develops a ring of off-white spores between the pre- andpost-N₂ treated growth. This sporulation is apparently triggered byoxygen stress.

Oxygen limitation, as well as glucose limitation, also triggerssporulation of Thielavia in submerged culture. In fact, such sporulationoccurs spontaneously after 3-4 days in ASPO4 medium with 20 g/l glucose.However, under fermentation conditions, this sporulation is undesirable,as the mycelial biomass rapidly disappears and is replaced by spores,thereby reducing productivity. To overcome this problem, anonsporulating strain is created from Thielavia terrestris E373. A shakeflask with 10⁶ UV-exposed (30 seconds exposure leading to 40% kill)spores/ml of medium (ASPO4 with 2 g/l glucose) is cultivated for 5 daysat 42° C. Mycelium for this culture is diluted in 0.1% Tween solutionspread on PDA plates, and incubated 24 hours at 42° C. The eight largestcolonies are picked, based on the assumption that mycelium fragmentswill grow faster than spores, which must first germinate. The selectedcolonies are transferred to shake flasks; two out of the eight selecteddo not sporulate at all in these submerged cultures, while the other sixshow some degree of sporulation.

Chosen for initial study in tank fermentation are Thielavia terrestris(strains E373 and ATCC 20627), Myceliophthora thermophila (strain A421),Sporotrichum cellulophilum (strain ATCC 20493), and Thermoascusthermophilus (strains 2050 and CBS 759.71), Mucor pusilus (strain A209),Acremonium alabamense (A2082), Talaromyces emersonii (strain A577).Strains designated "A" are available in the culture collection of NovoNordisk A/S, Bagsv.ae butted.rd, Denmark; strains designated "E" areavailable in the culture collection of Novo Nordisk Entotech, Davis,Calif.

II. Fermentor Evaluation

The medium used in tank fermentation is as follow:

    ______________________________________                                                            Batch                                                     ______________________________________                                        MgSO.sub.4.7H.sub.2 O 2     g/l                                               KH.sub.2 PO.sub.4     5     g/l                                               Citric acid.1H.sub.2 O                                                                              4     g/l                                               Yeast extract         10    g/l                                               NH.sub.4 sulfate      10    g/l                                               CaCl.sub.2            2     g/l                                               AMG trace metals*     0.5   ml/l                                              Pluronic              1     ml/l                                              ______________________________________                                         *contains ZnSO.sub.4.7H.sub.2 O 14.3 g/l; CuSO.sub.4 5H.sub.2 O 2.5 g/l;      NiCl.sub.2.6H.sub.2 O 0.5 g/l; FeSO.sub.4.7H.sub.2 O 13.8 g/l;                MnSO.sub.4.H.sub.2 O 8.5 g/l; citric acid 3.0 g/l                        

Tap water is used, and pH is adjusted to 6 before autoclaving.

Carbon source: 50% glucose added to 100 g/l based on initial volume (2l)

Fermentation is conducted at 42°-46° C., at pH 5.0, +/-0.1, adjustedwith H₃ PO₄ or NaOH. Applicon fermentors with increased impeller sizeare used. The concentration of dissolved oxygen (DOT) is kept >20% ofsaturation concentration with an aeration rate of 1 volume per volumeper minute and agitation speed between 800-1400 rpm.

Biomass concentration is estimated by dry cell weight. Twenty mls ofcultures are filtered through preweighed 20 μm membranes. Filtercakesare washed twice with H₂ O, dried 48 hours at 96° C. and weighed.Viscosity is determined with a Bohlin Reologi, Inc. "Visco 88" which isequipped with the "C14" cup-and cylinder system. The system switch isset to "1" and the speed is set to "8". This turns the cylinder withinthe cup at 1000 rpm and delives a shear rate of 1222/s. Whole culturesamples are removed from fermentors and measured within two minutes onroom temperature equipment. Approximately 10 mLs of sample is put intothe cup which in turn is fixed onto the Visco 88 with the cylinder fullysubmerged. The initial viscosity reading, produced within seconds of thestart of cylinder rotation, is recorded.

Six fermentation runs of T. terrestris E373 and ATCC 20627, M.thermophila 421, S. cellulophilum ATCC 20493, T. thermophilus A2050 andA. oryzae A1560 (control) are analyzed for viscosity. All runs are 100g/l glucose batch fermentations at pH 5 and 1100 rpm. The thermophilicstrains are grown at 42° C. and A. oryzae is grown at 37° C. The dataare presented in Table 1, showing viscosity values in pascal; the lowerthe pascal value, the less viscous the culture. The measured viscositydata are for fresh samples measured during the first seconds of testingin the rheometer. The extrapolated viscosity data, comparing the strainsat 30 g/l biomass are shown in FIG. 1.

                  TABLE 1                                                         ______________________________________                                        Comparative viscosities of thermophiles                                                    TIME      BIOMASS   VISCOSITY                                    STRAIN       (h)       (g/l)     (pascal)                                     ______________________________________                                        Aspergillus  24        34.1      0.473                                        oryzae, A1560                                                                              43.5      36.9      0.399                                                     51.5      27.6      0.217                                        Thielavia    24        28.2      0.028                                        terrestris, E373                                                                           43.5      33.5      0.013                                        Myceliophthora                                                                             24        20.8      0.045                                        thermophila, A421                                                                          43.5      30.3      0.094                                                     51.5      27.8      0.035                                        Sporotrichum 17.5      22.2      0.065                                        cellulophilum,                                                                             24        30.0      0.096                                        ATCC 20493   40        46.8      0.144                                                     47        52.9      0.178                                        Thielavia    24        6.1       0.018                                        terrestris, ATCC                                                                           40        51.1      0.016                                        20627                                                                         Thermoascus  40        17.4      0.025                                        thermophilus,                                                                              47        22.4      0.037                                        A2050        71.5      31.6      0.042                                                     112       36.6      0.053                                        ______________________________________                                    

As the data show, by far the most viscous strain is the control A.oryzae strain. The least viscous strains are the two Thielavia strains.These strains are observed to be easily mixed and aerated in thefermentors and show a very homogeneously dispersed mycelium, which formsa loose structure of closely associated, highly branched mycelia thatcontinuously break up during growth. Neither big pellets nor aggregatesare formed.

The Myceliophthora strain is very similar to Thielavia, but gives anelongated and less branched growth form, resulting in somewhat greaterviscosity. Measurements on Acremonium are not obtained, but visualobservation indicates that it shows a morphology and viscosity similarto Thielavia. Thermoascus also shows good morphology, similar toThielavia. Sporotrichum is also good morphologically, but produces largeamounts of a green pigment and starts to lyse under these fermentationconditions. Mucor pusilus produces a less than optimum morphology, withone large cake of mycelia, and is not analyzed further. Talaromyces alsois not taken further because it produces higher amounts of proteaseactivity than the other strains. Table 2 illustrates data for growthrates, measured at biomass concentrations of 2-15 g/l, and for proteaseproduction for the strains tested in the fermentor. Growth rate isestimated from the growth curve, where an almost linear biomass increaseis seen from 2-15 g/l dry biomass concentration(biomass is determined byfiltration, drying and weighing remaining mycelium on the filter.

                  TABLE 2                                                         ______________________________________                                        Growth rate and protease formation in                                         glucose batch fermentations at pH5 and 42° C.                                       GROWTH RATE                                                                   RELATIVE    PROTEASE                                             STRAIN       TO A. ORYZAE                                                                              PRODUCTION                                           ______________________________________                                        Thermoascus  1 X         low protease                                         thermophilus                                                                  Mucor pusilus                                                                              2.5 X       very high protease(all)                              Myceliophthora                                                                             1 X         very high alkaline                                   thermophila              protease                                             Thielavia terrestris                                                                       3 X         high acid protease                                   Acremonium alabamense                                                                      3 X         high acid protease                                   Talaromyces emersonii                                                                      1 X         very high protease(mainly                                                     acidic)                                              Sporotrichum 2 X         some protease(mainly                                 cellulophilum            acidic)                                              ______________________________________                                    

Another method for evaluating fungal morphology and to determine howsuitable it is for submerged tank fermentation is to attempt to obtainvery high biomass concentration. With unicellular organisms, such as E.coli and S. cerevisiae, it is possible to reach biomass concentrationclose to 100 g/l, but with fungi it is very difficult to reach a levelgreater than 75 g/l. The upper limit (caused by high viscosity and lackof oxygen transfer) for A. oryzae and A. niger is about 50-60 g/l.Thielavia terrestris E373 and the nonsporulating mutant described aboveare tested in submerged culture. Fermentation is at pH 5, 42° C. and inmedium that is double strength compared to earlier described batchmedium (200 g/l glucose). The nonsporulating mutant achieves a biomassof about 90 g/l after 140 hours (FIG. 3). This is an unusually highbiomass concentration in a fungal fermentation, and clearly demonstratesthe superiority of the strains having this type of morphology.

II. Expression of heterologous genes in thermophiles

A. Selectable marker vectors

The vector pJaL77 is used in transformation of host cells with thehygromycin B resistance selectable marker. This marker is based on theE. coli hygromycin B phosphotransferase gene, which is under the controlof the TAKA promoter. Briefly, the vector is constructed as follows. Thegene conferring resistance to hygromycin B is purchased from BoehringerMannheim as plasmid pHph-1. This gene is equipped with an ATG codon aswell as with suitable restriction sites at the amino and carboxy terminiby PCR, using the primers: 5'-GCT CAGAAGCTT CCATCC TAC ACC TCAGCA ATGTCG CCT GAA CTC ACC GCG ACG TCT-3' (N-terminal, SEQ ID NO:1) and 3'-CGTCCG AGG GCAAAG GAATAG CTCCAG AGATCT CAT GCT-5' (C-terminal, SEQ IDNO:2). The PCR fragment is cut with the restriction enzymes BamHI andXhoI and cloned into the corresponding sites in the Aspergillusexpression vector pToC68 (as described in WO 91/17243) to producepJaL77.

The plasmid pToC90 containing the amdS marker is constructed by cloninga 2.7 kb XbaI fragment from p3SR2 (Hynes et al. Mol. Cell. Biol. 3(8):1430-1439, 1983) into an XbaI cut and dephosphorylated pUC19 plasmid.

B. Expression vector

The vector pHD414 is a derivative of the plasmid p775(EP 238 023). Incontrast to this plasmid, pHD414 has a string of unique restrictionsites between the TAKA promoter and the AMG terminator. The plasmid isconstructed by removal of an approximately 200 bp long fragment(containing undesirable RE sites) at the 3' end of the terminator, andsubsequent removal of an approximately 250 bp long fragment at the 5'end of the promoter, also containing undesirable sites. The 200 bpregion is removed by cleavage with NarI (positioned in the pUC vector)and XbaI (just 3' to the terminator), subsequent filling in thegenerated ends with Klenow DNA polymerase+dNTP, purification of thevector fragment on a gel and religation of the vector fragment. Thisplasmid is called pHD413. pHD413 is cut with StuI (positioned in the 5'end of the promoter) and PvuII (in the DUC vector), fractionated on geland religated, resulting in pHD414. A strain of E. coli containing theapproximately 1,100 bp xylanase HindII/XbaI cDNA fragment in pYES isdeposited in DSM as DSM 6995. The xylanase cDNA fragment is isolatedfrom one of the clones by cleavage with HindIII/XbaI. The fragment ispurified by agarose gel electrophoresis, electroeluted, and made readyfor ligation reactions. The cDNA fragment is ligated into pHD414 toproduce pAXX40-1-1, which is deposited as NRRL B-21164. The xylanasegene is deposited as DSM (Deutsche Sarmmlung Von Mikrooroganismen undZellkulturen GmbH) 6995.

III. Transformation of thermophilic hosts

The following general procedures are used in transformation of all theStrains tested, with exceptions noted expressly:

100 ml of MY51 medium (maltodextrin, 30 g/l; MgSO₄.7H₂ O, 2 g/l; K₂ PO₄,10 g/l, K₂ SO₄, 2 g/l; citric acid, 2 g/l; yeast extract, 10 g/l; AMGtrace metals, 0.5 ml; urea 1 g/l; (NH₂)SO₄, 2 g/l, pH 6.0) is inoculatedwith mycelial plugs (2 cm diameter) of the strain to be transformed andincubated with shaking at 42° C. for 14 hours. The mycelium is harvestedby filtration through miracloth and washed with 200 ml of 0.6M MgSO₄.The mycelium is suspended in 15 ml of 1.2M MgSO₄, 10 mM NaH₂ PO₄,pH=5.8. The suspension is cooled on ice and 1 ml of buffer containing120 mg of Novozyme® 234 is added. After 5 minutes, 1 ml of 12 mg/ml BSA(Sigma type H25) is added and incubation with gentle agitation continuedfor 1-3 hours, depending on the strain, at 30° C. until a large numberof protoplasts are visible in a sample inspected under the microscope.For Acremonium and Thielavia, protoplasting efficiency is relativelylow, and these strains require longer incubation periods (2-3 hours)until sufficient protoplasts are obtained for transformation.

The suspension is filtered through miracloth, the filtrate istransferred to a sterile tube and overlaid with 5 ml of 0.6M sorbitol,100 mM Tris-HCl, pH=7.0. Centrifugation is performed for 15 minutes at2500 rpm and the protoplasts are collected from the top of the MgSO₄cushion. Two volumes of STC (1.2M sorbitol, 10mM Tris-HCl pH=7.5, 10 mMCaCl₂) are added to the protoplast suspension and the mixture iscentrifuged for five minutes at 2500 rpm. The protoplast pellet isresuspended in 3 ml of STC and repelleted. This is repeated, and thenthe protoplasts are resuspended in 0.2-1 ml of STC.

100 μl of protoplast suspension is mixed with 5-25 μg of the appropriateDNA in 10 μl of STC. Each strain is cotransformed with pAXX40-1-1 and aplasmid containing a selectable marker. Plasmids pToC90 contains the A.nidulans amdS gene, and is used for transformation and selection forgrowth on acetamide as the sole nitrogen source. Plasmids pJaL77 is usedfor transformation and selection of resistance to hygromycin B (150μg/ml).

The mixtures are left at room temperature for 25 minutes. 0.2 ml of 60%PEG 4000 (BDH 29576), 10 mM CaCl₂ and 10 mM Tris-HCl pH=7.5 is added andcarefully mixed twice and finally 0.85 ml of the same solution is addedand carefully mixed. The mixture is left at room temperature for 25minutes, spun at 2500×g for 15 minutes and the pellet resuspended in 2ml of 1.2M sorbitol. After one more sedimentation the protoplasts arespread on the appropriate plates. Protoplasts are spread on minimalplates (Cove, Biochem. Biophys. Acta 113: 51-56, 1966) containing 1.0Msucrose, pH=7.0, 10 mM acetamide as nitrogen source (when amdS is theselection marker) and 20 mM CsCl to inhibit background growth. Themedium differs when hygB is the selection marker in the use of 10 mMsodium nitrate as nitrogen source, and the presence of 150 μg/mlhygromycin B. The selection plates are incubated at 42° C. for 5 days.All the transformants which grow on COVE selection medium aretransferred to COVE II (COVE medium without calcium chloride and with asucrose concentration of 30 g/l) plates containing AZCL-xylan (0.2%) andthe respective selection agent. Cotransformants are identified by therapid formation of a blue halo in and around the fungal colony. Theresults of transformation experiments and the number of cotransformantsidentified are given in Table 3.

                  TABLE 3                                                         ______________________________________                                        Results of transformation of thermophiles                                              SELEC-  NUMBER OF     NUMBER OF CO-                                  STRAIN   TION    TRANSFORMANTS TRANSFORMANTS                                  ______________________________________                                        Myceliophthora                                                                         amdS    42            18                                                      HygB    0             0                                              Sporotrichum                                                                           amdS    35            15                                                      HygB    0             0                                              Thielavia                                                                              amdS    18            4                                                       HygB    0             0                                              Acremonium                                                                             amdS    23            10                                                      HygB    0             0                                              ______________________________________                                    

Hygromycin selection is not successful in any of the thermophilic fungiinvestigated presumably because the promoter is not efficient in thesestrains. However, with amdS selection, transformants are obtained in allstrains except Thermoascus. Co-transformation frequency is between20-40%.

D. Evaluation of xylanase expression in transformants

All the co-transformants identified by xylanase-plate assays aresubjected to shake flask evaluation for xylanase productivity. M401medium of the following composition (g/l) is used: maltodextrin, 50.0;MgSO₄.7H₂ O, 2.0; KH₂ PO₄, 2.0; citric acid, 4.0; yeast extract, 8.0;AMG trace metal solution, 0.5 ml; ammonium sulfate, 2.0; urea, 1.0. Thecultures are incubated at 42° C. and xylanase activity is measured everyday starting from 24 hours. Xylanase activity in culture broths isdetermined using 0.2% AZCL-xylan (Megazyme Co. Australia) suspended in acitrate phosphate buffer, pH 6.5. The culture fluid is diluted, usually100-fold, and 10 μl of diluted culture fluid is mixed with 1 ml of 0.2%AZCL-xylan substrate. The mixture is incubated at 42° C. for 30 minutes.The reaction mixture is mixed well every 5 minutes. At the end ofincubation, the undigested substrate is precipitated by centrifugationat 10,000 rpm for 5 minutes. The blue dye released from this substrateis quantified by absorbance at 595 nm and the amount of enzyme activityin the culture broths is calculated from a standard made with an enzymepreparation with known activity. An endoxylanase unit (EXU) isdetermined relative to an enzyme standard prepared under identicalconditions. Untransformed strains are also grown under identicalconditions and compared with the transformants.

The data presented in FIG. 2 (based on the peak of activity) indicatethat all untransformed strains produce xylanase activity at very lowlevels, while some of the transformants produce up to 5-10 fold greateractivity than the untransformed strains. SDS-PAGE analysis of the spentculture medium reveals the presence of a 22 kD Humicola xylanase proteinband only in the transformants, although at very low levels. Thisillustrates the potential for expression of heterologous genes inthermophilic fungi. The order of productivity of xylanase is in theorder of Myceliophthora-Sporotrichum-Acremonium-Thielavia.

E. Confirmation of Transformation and integration of expression vectors

To unequivocally demonstrate the transformation and integration ofexpression vectors, southern hybridization analyses are performed usingtotal DNA isolated from untransformed and selected transformants fromeach strain. Two best transformants from each strain are selected forsouthern hybridization analyses. Total genomic DNA isolated from thesestrains is digested with EcoRI, DNA fragments are separated therough 1%agarose gel and blotted. Since Thielavia and Acremonium represent theperfect and imperfect stages of the same strain, only DNA fromAcremonium is used for the hybridization experiments. First, the blot isprobed with the Aspergillus nidulans amdS gene, the selection markerused for transformation. The results show the presence of amdS gene onlyin the transformants but not in the untransformed negative controls.Reprobing of the same blot, after stripping off the amdS probe, with a1.2 kb HindIII and XhoI fragment of Humicola insolens xylanase cDNA,show the following: Only the transformants of Acremonium but not theuntransformed strain shows the presence of H. insolens cDNA hybridizingbands. The untransformed as well of transformants of Myceliophthora andSporotrichum show the presence of an about 2 kb band hybridizing to theprobe (possibly indicating the presence of DNA sequences in thesestrains sharing homology to the H. insolens xylanase gene), while thetransformants show additional high molecular weight bands hybridizing tothis probe.

What we claim is:
 1. A recombinant fungal host strain in which saidstrain is obtained from a member of the genus Acremonium, Corynascus,Thielavia, Myceliophthora, Thermoascus, Sporotrichum, Chaetomium,Ctenomyces, or Scytalidium and in which said strain produces at least 75g/l of biomass concentration, dry weight, in tank fermentation.
 2. Thestrain of claim 1 which is obtained from Thielavia terrestris,Acremonium alabamense, Myceliophthora thermophilum, or Sporotrichumcellulophilum.
 3. The strain of claim 1 which is obtained from anonsporulating mutant of Thielavia.